Wnt proteins comprise a family secreted signaling molecules that play major roles in tissue development and cell fate determination during embryogenesis, as well as tissue maintenance and oncogenesis in adults. In order to signal correctly, Wnt proteins need to be processed, modified and secreted. Wnt processing involves the attachment of two fatty-acyl moieties, palmitate and palmitoleate. Inability to incorporate these fatty acids renders Wnt unable to initiate the intracellular signaling cascade or to be efficiently secreted. Porcupine (Porcn), a multipass transmembrane protein, is the acyltransferase responsible for attachment of these fatty- acid adducts. Porcn has been postulated as an appealing target for the development of inhibitors that could modulate Wnt signaling activity in Wnt-related diseases such as cancer and skeletal abnormalities. Unfortunately, the limited knowledge about Porcupine's biochemical and functional properties has hampered this goal. The long-term objective of this project is to characterize the mechanism by which Porcupine recognizes its substrates (palmitate and Wnt) and catalyzes the transfer of fatty acids onto Wnt. While performing preliminary studies, I discovered that Porcn itself is palmitoylated. I will follow up on this novel finding by determining the functional significance of Porcn palmitoylation. Experiments in Aim 1 will ascertain whether palmitate incorporation into Porcn represents formation of an acyl-enzyme intermediate or is the result of modification by another palmitoyl acyltransferase. The site(s) of palmitoylation on Porc will be identified and mutated and effects on Porcn stability, localization and acyltransferase activity will be monitored. Specific Aim 2 involves a comprehensive structure-function analysis of Porcn, using a mutagenesis approach, to identify key residues required for fatty acid binding, Wnt binding and palmitoyl acyltransferase activity. The functional relevance of these residues will be determined by analyzing the effect of point mutations on Porcupines' biochemical properties and activity, and on Wnt secretion and activity. In addition, the membrane topology and oligomerization state of Porcn will be determined. This approach will allow us to determine where the catalytic residues lie within the plane of the surrounding lipid bilayer, as well as to map the predicted transmembrane domains. These studies will contribute to our knowledge of a relatively unexplored multipass membrane protein that regulates a critical family of signaling molecules. PUBLIC HEALTH RELEVANCE: Elevated levels of the protein Wnt have been linked to a wide variety of human cancers (breast, colon, lung), as well as in human skeletal abnormalities. In order for Wnt to function correctly, the enzyme Porcupine needs to add two fatty acids (palmitate and palmitoleate) onto Wnt. The goal of this project is to decipher the mechanism by which Porcupine acylates Wnt, as well as to investigate Porcupine's possible role as an oncogene and potential target for the development of selective inhibitors.